The CAPRISA Immunology Core will perform viral characterization, humoral and cellular immunity assays, HLA genotyping and" macrophage resistance assays in three separate laboratories at the National Institute for Virology in Johannesburg and the HIV-1 Molecular Virology laboratory at the Africa Center/University of Natal in Durban. This core will meet the immunological needs of two CAPRISA projects; the acute seroconvertor project and the project involving highly exposed persistently seronegative individuals. To perform the assays needed by both projects, the core has four components: Viral characterization and humoral immunity laboratory led by Core Leader, Lynn Morris; Cellular immunity laboratory led by Core co-Leader, Clive Gray; HLA genotyping laboratory led by Core co-Leader, Adrian Puren; and Macrophage function laboratory led by Core co-Leader, Sharon Cassol. The humoral immunity laboratory will be involved in virus isolation and characterization as well as an assessment of neutralizing antibody activity to both autologous and heterologous viruses. Envelope genes of isolates of interest will be cloned and used in transient expression systems to more clearly define their activity. Envelope genes will be sequenced to determine what genetic changes are associated with changes in co-receptor usage or resistance to virus neutralization. The cellular immunity laboratory will establish methodologies to assess the breadth and magnitude of CD4+ and CD8+ T cell responses. Gamma interferon ELISPOT assays will be used from freshly isolated PBMC to screen for peptide and optimal epitope responses. Flow cytometry (FACS) will be used to identify specific CD4+ and CD8+ T cell IFN-g responses to either pools or single peptides. Simultaneous DNA ploidy analysis on gated CD4+ T cells will be used to assess T helper cell responsiveness to a range of HIV-1 subtype C-derived proteins and peptides. T cell lines and clones will be grown in the cellular immunity laboratory and used to map CTL epitopes. To achieve CTL epitope mapping, all B cell lines will be HLA genotyped at high resolution by the HLA laboratory. The HLA laboratory will assist in defining the HLA-restriction and identifying epitopes, performing typing at both low and high resolution by sequence specific primer (SSP)-PCR and automated sequencing, respectively. The macrophage function laboratory in Durban will work closely with the cellular immunity laboratory at the National Institute for Virology to examine the role of macrophages in resistance to HIV-1 infection. Activation and functional status of monocyte-derived macrophages and dendritic cells will be assessed. Cytokine/chemokine expression patterns will be examined by flow cytometry, RNase protection, ELISA and cytology. Activation of signaling cascades will be examined. PCR-based assays will be performed to determine if pre-integration or integrated DNA, or early and late mRNA transcripts are present in macrophages. The CAPRISA investigators involved in this core will also participate in the training program as outlined in the administration core. In this regard, the core will work in collaboration with three US laboratories based at the University of Washington (Julie McElrath) on the highly exposed persistently seronegative individuals project and Duke University on humoral immunity assays (David Montefiori) and CTL assays (Guido Ferrari). The assays undertaken by the four components in this core will determine which responses correlate with viral control and viral escape in the acute seroconvertor project and potential reasons for the phenomenon of immunological resistance to HIV-1 in the highly exposed persistently seronegative individuals project.